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Standard reference ranges for all laboratory test values are mandatory. This study was designed to establish a reference range for blood vitamin B1 levels, since the normal range has not been determined in the Japanese population. We founded the Japan Committee for Vitamin Laboratory Standards, which was incorporated with the Vitamin Society of Japan and the Japanese Society of Nutrition and Food Science. We standardized whole blood vitamin B1 levels using three HPLC techniques (post-column reverse-phase HPLC, pre-column reverse-phase HPLC, and precolumn GP-HPLC). The reference range was obtained in 54 volunteers administered a 1,800 kcal diet with 2 mg of vitamin B1 (1.74 mg measured) daily to avoid marginal vitamin B1 deficiency in the population. The range for each assay was 26-47, 28-51, and 28-56 ng/ml, respectively. Our data suggest that 26-28 ng/ml is the lower limit of normal for whole blood vitamin B1, but further studies in a larger population are needed in order to obtain more definitive results.

TCR-gamma delta-bearing T cells have been reported to be increased in several immunodeficient patients. However, their functional role and phenotypic characterization have not yet been well documented. In this study we examined the surface phenotypes and functional properties of TCR-gamma delta+ cells from several patients with primary immunodeficiency syndrome. It was demonstrated that TCR-gamma delta+ cells detected by TCR-delta 1 mAb were increased in some of the patients, particularly in patients with Wiskott-Aldrich syndrome and severe combined immune deficiency. The TCR-gamma delta+ cells showed such a unique profile that more than 60% of the cells expressed delta-TCS1, which is normally present in a lesser amount, and that most of the cells lacked CD5 T lineage marker. TCR-gamma delta+ cells from the patients with primary immunodeficiency syndrome served as NK cells as observed in normal individuals, while displaying weak LAK and allogeneic cell-specific killer activities. The TCR-gamma delta+ cells were classified into several subpopulations according to their antigenic phenotype, then their NK activity of normal individuals and patients, lymphokine-activated killer and allo-specific killer activities of normal individuals were compared among the subpopulations. Delta-TCS1+ cells mediated almost the same killer activities as total TCR-gamma delta+ cells, whereas CD8+ TCR-gamma delta+ cells displayed stronger cytotoxic activities in both normal subjects and the patients with primary immunodeficiency syndrome.

We report here that soluble CD40 ligand (sCD40L) is released from human platelets when activated with collagen or thrombin. The sCD40L was detectable in the culture supernatants of platelets within 30 min after stimulation in vitro, and reached maximal levels in 3 h. The release was blocked by the metalloproteinase inhibitor, KB8301, indicating that the soluble CD40L is made by cleaving the membrane bound CD40L expressed on activated platelets. The sCD40L was undetectable in the supernatant of the activated platelets obtained from patients with X-linked hyper IgM syndrome (XHIM), who have defects in CD40L gene. Since sCD40L has been shown to have biologic function on the activation of vascular endothelial cells and B cells, these findings suggest that platelets play some roles in both inflammation and humoral immune response by releasing soluble CD40L.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, immunodeficiency, and eczema. X-linked thrombocytopenia (XLT) is a mild form of WAS with isolated thrombocytopenia. Both phenotypes are caused by mutation of the Wiskott-Aldrich syndrome protein (WASP) gene. In this study, we identified mutations of the WASP gene in 10 Japanese patients from 9 unrelated families with WAS/XLT. All XLT patients (n = 3) and one WAS patient had a missense mutation at the PH domain of WASP. Two WAS patients had nonsense mutations. One WAS patient had exon 8 skipping caused by one nucleotide deletion at the acceptor site of intron 7. Three WAS patients had genomic deletions; one of the three had a large genomic deletion involving exons 3 to 7. Codons 45 and 86 seem to be the hot spots of the WASP mutation, because missense mutations in these codons have been reported previously in several WAS/XLT patients in addition to the patients in this report, and patients with the same mutation show a similar clinical phenotype. All other mutations are novel, indicating that the mutations of WASP are heterogeneous. EB virus-transformed cell lines from XLT patients expressed nearly normal amounts of WASP, whereas those from typical WAS patients expressed almost undetectable amounts of WASP. We conclude that the analysis of gene mutation and protein expression of WASP are useful together in assessing the severity of WAS.

A cDNA with homology to fatty acid desaturases was selected by searching the cDNA data bank of Dictyostelium discoideum (http://www. csm.biol.tsukuba.ac.jp/cDNAproject.html) with conserved histidine box motifs. Using this sequence, genomic DNA encoding the Delta5 desaturase was amplified from the genomic DNA of D. discoideum, and its desaturase activity was confirmed by the overexpression mutation in D. discoideum and the gain-of-function mutation in yeast. The cloned cDNA is 1565 nucleotides in length, and the deduced amino-acid sequence comprised 467 amino-acid residues containing an N-terminal cytochrome b5 domain that shared 43% identity with cytochrome b5 of Oryza sativa. The whole sequence was 42% identical to the Delta5 desaturase of Mortierella alpina. This desaturase is a novel member of the cytochrome b5-containing Delta5 fatty acid desaturase. As we have already reported one other Delta5 desaturase in Dictyostelium, this organism is the first to be confirmed as having two functional Delta5 fatty acid desaturase genes. The substrate specificities of the two functional Delta5 desaturases of D. discoideum were also examined.

Standard reference ranges for all laboratory test values are mandatory. This study was designed to establish a reference range for blood vitamin B1 levels, since the normal range has not been determined in the Japanese population. We founded the Japan Committee for Vitamin Laboratory Standards, which was incorporated with the Vitamin Society of Japan and the Japanese Society of Nutrition and Food Science. We standardized whole blood vitamin B1 levels using three HPLC techniques (post-column reverse-phase HPLC, pre-column reverse-phase HPLC, and precolumn GP-HPLC). The reference range was obtained in 54 volunteers administered a 1,800 kcal diet with 2 mg of vitamin B1 (1.74 mg measured) daily to avoid marginal vitamin B1 deficiency in the population. The range for each assay was 26-47, 28-51, and 28-56 ng/ml, respectively. Our data suggest that 26-28 ng/ml is the lower limit of normal for whole blood vitamin B1, but further studies in a larger population are needed in order to obtain more definitive results.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, immunodeficiency and eczema. X-linked thrombocytopenia (XLT) is a mild form of WAS with isolated thrombocytopenia. Both phenotypes are caused by mutation of the Wiskott-Aldrich syndrome protein (WASP) gene. In this study we investigated the role of WASP in the differentiation of CD34-positive (CD34+) cells isolated from the bone marrow of patients with WAS (n = 5) or with XLT (n = 4). Megakaryocyte colony formation was significantly decreased in patients with WAS when compared with normal controls. The formation of granulocyte-macrophage colonies and erythroid bursts were also decreased in WAS patinets. In contrast, in XLT patients, formation of all these colonies was normal. However, in vitro proplatelet formation of megakaryocytes induced by thrombopoietin was markedly decreased in both XLT and WAS. Electron microscopic examination revealed that megakaryocytes obtained from WAS or XLT patients grown in vitro had abnormal morphologic features, which seemed to be caused by defective actin cytoskeletal organization, including labyrinth-like structures of the demarcation membrane system and deviated distribution of the alpha-granules and demarcation membrane system. These observations indicate that WASP is involved in the proliferation and differentiation of CD34+ haemopoietic progenitor cells probably by its participation in signal transduction and in the regulation of the cytoskeleton.

A 39-year-old female suffered from diffuse goiter, palpitation, finger tremor and body weight loss for about one year. Then she developed acute onset of myalgia and swelling of calves, and muscle weakness of proximal limbs. She could not walk because of myalgia and muscle weakness, and was admitted to our hospital 4 days after the onset of muscle symptoms. On admission, her pulse was 110 per minute and she had finger tremor of 11-12 Hz. The thyroid gland was markedly and diffusely enlarged with an elastic soft surface. She presented muscle weakness of proximal limbs and neck, and had intermittent swelling and myalgia on calves. Deep tendon reflexes were increased in all extremities. The erythrocyte sedimentation rate was 22 mm per hour. Eosinophilia was not recognized. Serum CK level was elevated to 671 IU/l. Serum free T3 was higher than 21.7 pg/ml and free T4 was also elevated to 10.19 ng /dl. Serum TSH was lower than 0.05 microU/ml and thyroid stimulating antibody was 1,302.0%. Muscle biopsy of her left gastrocnemius muscle revealed markedly hypertrophic fascia with inflammatory cellular infiltration on HE staining. Inflammatory change was also recognized in muscle tissue and in perivascular region of perimysium. Variation of fiber size, necrotic fibers, and central nuclei were also seen. From these clinical and laboratory findings she was diagnosed as having Basedow's disease associated with fasciitis and polymyositis. Her thyroid function was improved by anti-thyroid drug, and swelling and myalgia of sural regions and weakness of proximal limbs were also improved by steroid therapy. Only one case of Basedow's disease associated with fasciitis and seven cases of that associated polymyositis have so far been reported. This is the first case report of fasciitis associated with Basedow's disease and polymyositis.

A 7 month old Japanese boy was diagnosed to have Wiskott-Aldrich syndrome (WAS) because of eczema, thrombocytopenia, progressive immune defect and CD43 (sialophorin) abnormality. He had developed repeated infections since 16 months of age. Gamma delta T cell-receptor positive T cells in the peripheral blood were gradually increased from 3.1% (7 months of age) to 5.6% (12 months), 19.6% (18 months) and 56.7% (25 months). The phenotypes of expanded gamma delta T cells were delta TCS1-positive (V delta 1-J delta 1/J delta 2) and CD8 dim-positive. The proportion of increased granular lymphocytes correlated well with that of gamma delta T cells. The significance of peripheral expansion of gamma delta T cells and granular lymphocytes in WAS is discussed.

A patient with severe combined immunodeficiency was transplanted with T cell depleted haploidentical bone marrow from his father and was later given a thymic graft from an unrelated donor. alpha/beta and gamma/delta T cells of bone marrow donor origin appeared only after the thymus transplantation procedure. Among the peripheral blood lymphocytes (PBL), gamma/delta T cells comprised 10-20% and most of them were delta TCS1+. The alpha/beta T cells were single positive cells, either CD4+ or CD8+. Expression of CD5, CD7 and CD8 alpha,beta molecules on alpha/beta T cells was reduced. Functional studies showed that gamma/delta T cells proliferated slightly in response to anti-CD3 stimulation, and proliferated well with exogenous IL-2 stimulation, while alpha/beta T cells did not proliferate following mitogenic stimulation even in the presence of IL-2. gamma/delta T cells but not alpha/beta T cells exhibited some LAK activity after culturing with IL-2. Since alpha/beta T cells expressed IL-2R alpha and beta chains after mitogenic stimulation and bound IL-2, the deficit(s) in these cells was considered to occur after IL-2 binding to the IL-2R. These results indicate thymic dependency of both types of T cells and that two types of T cells differed in the acquisition of IL-2 responsiveness during development.

Two adult patients with acute mixed lineage leukemia (AMLL) having combined Philadelphia chromosome (Ph1) positivity and monosomy 7 are presented. The phenotypes of leukemic blasts from both cases were almost same (early B-lymphoid lineage and myeloid lineage); CD10+, CD13+, CD19+. HLA-DR+, and dual-color analysis showed simultaneous expression of CD10 (CD19) and CD13 antigens in individual blasts (biphenotypic) in both cases. On molecular analysis, the leukemic blasts showed rearrangement in the first intron of the BCR gene with breakpoint just outside of 3' end of m-BCR-2 (bcr 3) in case 1, and in the M-BCR in case 2. Immunoglobulin heavy chain gene (IgH) rearrangement was noted in both cases, but rearrangement of the T-cell receptor beta-chain gene (TCR beta) was detected only in case 1. Clinically, both cases achieved complete remission by the combination chemotherapy consisting of L-asparaginase, doxorubicin, vincristine, and prednisolone (L-AdVP). In remission, all these molecular abnormalities disappeared in both patients. These results suggest that the Ph1-positive and monosomy 7 AMLL in adults is de novo acute leukemia with both early B-lymphoid and myeloid phenotypes and may arise from malignant transformation of pluripotent stem cell, and expresses a heterogenous rearrangement pattern of the BCR gene.

Between 1983-1988 bone marrow samples obtained from 195 peroxidase-negative leukemia patients were analyzed for their surface antigens. Thirteen of these patients (6.7%) had myelomonocytic-positive and lymphoid-negative antigens. These leukemic cells reacted with CD13 in eight patients, CD33 in seven, CD11 in six and CDw41 in two. In none of these patients did the leukemic cells react with CD1, CD2, CD3, CD4, CD5, CD8, CD10, CD19 or CD20. Leukemic cells from two patients were reactive with CD7. These leukemic cells demonstrated L2 morphology in 11 patients and L1 morphology in one patient. The leukemic cells from the final patient were diagnosed as those of leukemic transformation of myelodysplastic syndrome. Chromosomal abnormality was observed in approximately half of the patients examined (6/10). Cytochemical analysis revealed that the leukemic cells were negative for periodic acid Schiff stain but positive for acid phosphatase. The prognosis of these patients was markedly poor as compared to acute lymphocytic leukemia or typical peroxidase-positive nonlymphocytic leukemia. Complete remission was induced in only 30% of patients and duration of survival was short (4.7 months). This suggests that myelomonocytic antigen-positive peroxidase-negative acute leukemia is a distinct type of leukemia and may require more aggressive therapy to improve survival.

TCR-gamma delta-bearing T cells have been reported to be increased in several immunodeficient patients. However, their functional role and phenotypic characterization have not yet been well documented. In this study we examined the surface phenotypes and functional properties of TCR-gamma delta+ cells from several patients with primary immunodeficiency syndrome. It was demonstrated that TCR-gamma delta+ cells detected by TCR-delta 1 mAb were increased in some of the patients, particularly in patients with Wiskott-Aldrich syndrome and severe combined immune deficiency. The TCR-gamma delta+ cells showed such a unique profile that more than 60% of the cells expressed delta-TCS1, which is normally present in a lesser amount, and that most of the cells lacked CD5 T lineage marker. TCR-gamma delta+ cells from the patients with primary immunodeficiency syndrome served as NK cells as observed in normal individuals, while displaying weak LAK and allogeneic cell-specific killer activities. The TCR-gamma delta+ cells were classified into several subpopulations according to their antigenic phenotype, then their NK activity of normal individuals and patients, lymphokine-activated killer and allo-specific killer activities of normal individuals were compared among the subpopulations. Delta-TCS1+ cells mediated almost the same killer activities as total TCR-gamma delta+ cells, whereas CD8+ TCR-gamma delta+ cells displayed stronger cytotoxic activities in both normal subjects and the patients with primary immunodeficiency syndrome.

The blasts obtained from three freshly diagnosed acute myeloblastic leukemia (AML) patients were cultured in suspension to determine whether leukemic blast progenitors can indeed differentiate to form mature granulocytes. One patient was AML M2. The other two patients were bilineal and biphenotypic leukemia, respectively. Media conditioned by human bladder carcinoma line 5637 (5637-CM) or recombinant human granulocyte colony-stimulating factor (rhG-CSF) was added to stimulate growth. In suspension, clonogenic cells grew for 1-3 weeks in two patients, while they did not increase in one patient. After repeated subculture, cells of blast morphology decreased in percentage and polymorphonuclear neutrophils, eosinophils, and monocyte-macrophages appeared. Lymphoid cell component of the patient 2, who was diagnosed as bilineal leukemia by dual-color immunofluorescence analysis, decreased in number after suspension culture and cells of myeloid phenotype became dominant. The findings show that clonogenic blast progenitors can renew themselves and can also undergo terminal differentiation to mature end cells.

The objective of this study was to analyze the configuration of the lymphocytes in Wiskott-Aldrich syndrome (WAS) by studying the surface antigens from nine cases using dual-color immunofluorescence analysis. All the patients showed the increase of non-MHC-restricted cytotoxic cells, namely CD3+ WT31- delta TCS1+ (gamma/delta-T cell receptor (TCR)-bearing cells) and/or CD16+ natural killer cells. The gamma/delta-TCR+ cells of WAS, however, were unique since they did not express CD5, which is present on ordinary gamma/delta-TCR+ cells. A reduced number of CD4+ cells and an increased percentage of CD11b+ Leu7+ cells within a CD8+ subset were observed in all cases. With regard to B cell subpopulations, most cases showed reduced Fc epsilon R2-bearing B cells, despite an elevated serum IgE.